Our research focuses on events in the exocytotic pathway that occur in the highly specialized domain of the plasma membrane-cytoplasm interface. This region is superbly imaged by total internal reflection fluorescence microscopy (TIRFM), a technique that we use extensively in our research. In the previous grant period we developed a method that combines polarization and TIRFM (P-TIRFM) to image in real time topological changes in the plasma membrane with submicron spatial resolution. We can, for the first time, directly visualize topological features of the expanding fusion pore. The proposed studies will establish a framework for the understanding of the pathways taken by the granule membrane during and after fusion. Aim 1 will exploit the strengths of P-TIRFM as well as amperometry to study mechanisms underlying fusion pore expansion. We will study the regulation of this process by dynamin (preliminary results), synaptotagmin and granule contents (preliminary results). The dynamin studies will be empowered by the opportunity provided by Dr. Pietro De Camilli (Yale University) to investigate mouse chromaffin cells without dynamins 1 and 2 from genetically altered mice. Aim 2 is based upon our recent findings that 1) several granule membrane proteins remain colocalized for minutes in the plasma membrane, rather than dispersing and 2) clathrin puncta formation begins within 10 sec at fusion sites. We will investigate the novel concept that granule membrane proteins that remain colocalized in the plasma membrane after exocytosis (some of which bind clathrin adaptors) induce clathrin-mediated endocytosis (CME). Aim 3 will build upon the same preliminary results motivating Aim 2 and will determine the relationship between curvature and the progression of protein interactions in compensatory, clathrin-mediated endocytosis.